The primary goal of our research program is directed towards an understanding of the biosynthesis and function of D-alanyl-lipoteichoic acid (LTA), a macroamphiphile which plays a vital role in the growth and development of the bacterium. The chemical properties and cellular location of this polymer indicate that it functions in the assembly of the cell wall. The substitution of the hydrophilic moiety of LTA with D-alanine ester provided a mechanism for controlling the anionic charge. Our hypothesis is that the D-alanine esters of LTA play a role in determining cell shape and separation by regulating extramembranal cellular functions. Three areas of research will include: (a) establishment of the genetic organization of the operon for D-alanine ester formation; (b) delineation of the mechanism of D-alanine incorporation into LTA; (c) construction of defined genetic systems which can be used to effect changes in the D-alanine ester content. (a) The detailed characterization of the genetic components required for D-alanyl ester formation is a prerequisite for understanding LTA function. Thus, it is our aim to: (i) define the regulatory elements and limits of the operon; (ii) identify the genes of the operon; (iii) locate the gene for the D-alanine carrier protein (Dcp). (b) D-Alanine-Dcp ligase catalyzes the activation of D-alanine and its ligation to Dcp. Our goal is to understand the enzymic mechanism for the thiolesterification of the 4'-phosphopantetheine prosthetic group of Dcp with D-alanine. It is also our goal to identify the membrane proteins and carrier lipid that accept activated D-alanine from D-alanyl- Dcp. Because of the central importance of the carrier protein in the incorporation pathway, studies of its structure and function are necessary for identifying the specificity determinants that regulate its role as an acceptor and as a donor of activated D-alanine. (c) Based on the observations form part (a) and (b), our goal is to design isogenic mutants which will aid in the determination of the in vivo function of D-alanyl-LTA. These constructions will provide strains in which the LTA is either deficient or elevated in D-alanine ester content. The ability to modulate the ester content using genetically defined mutants will clarify the role of these esters in the determination of cell shape and separation. Each of the aims described in this proposal is designed to provide a detailed biochemical, genetic and structural understanding of D-alanyl- LTA biosynthesis. Achieving these goals will yield insights into the role of these macroamphiphiles in the physiology of the gram-positive bacterium. The elucidation of the D-alanine incorporation system will identity new targets which may be used for the rational design of antibacterial agents.